Covalent attachment of the hydrophilic polymer poly(ethylene glycol) ("PEG"), also known as poly(ethylene oxide) ("PEO"), to molecules and surfaces has important applications, including in biotechnology and medicine. In its most common form, PEG is a linear polymer having hydroxyl groups at each terminus: EQU HO--CH.sub.2 --CH.sub.2 O(CH.sub.2 CH.sub.2 O).sub.n CH.sub.2 CH.sub.2 --OH
This formula can be represented in brief as HO--PEG--OH where it is understood that --PEG-- represents the following structural unit: EQU --CH.sub.2 CH.sub.2 O(CH.sub.2 CH.sub.2 O).sub.n CH.sub.2 CH.sub.2 --
n typically ranges from approximately 10 to 2000.
PEG is commonly used as methoxy-poly(ethylene glycol), or mPEG in brief, in which one terminus is the relatively inert methoxy group, while the other terminus is a hydroxyl group subject to ready chemical modification: EQU CH.sub.3 O--(CH.sub.2 CH.sub.2 O).sub.n CH.sub.2 CH.sub.2 --OH mPEG
Similarly, other alkoxy groups such as benzyloxy and tert-butoxy can be substituted for methoxy in the above formula.
Branched PEGs are also commonly used. The branched forms can be prepared by addition of ethylene oxide to various polyols, including glycerol, pentaerythritol and sorbitol. For example, the four-armed branched PEG prepared from pentaerythritol is shown below: ##STR1## Branched PEGs can be represented as Q(--PEG--OH).sub.n in which Q represents a central core molecule such as pentaerythritol or glycerol, and n represents the number of arms which can range from three to a hundred or more. The hydroxyl groups are readily subject to chemical modification.
The copolymers of ethylene oxide and propylene oxide are closely related to PEG in their chemistry, and they can be substituted for PEG in many of its applications. EQU HO--CH.sub.2 CHRO(CH.sub.2 CHRO).sub.n CH.sub.2 CHR--OH R=H AND CH.sub.3
PEG is a useful polymer having the property of water solubility as well as solubility in many organic solvents. PEG is also non-toxic and non-immunogenic. When PEG is chemically attached to a water insoluble compound, the resulting conjugate generally is water soluble as well as soluble in many organic solvents. When the molecule to which PEG is attached is biologically active, such as a drug, this activity is commonly retained after attachment of PEG and the conjugate may display altered pharmacokinetics. For example, it has been demonstrated that the water insoluble antimalarial, artemisinin, becomes water soluble and exhibits increased antimalarial activity when coupled to PEG. See Bentley et al., Polymer Preprints, 38(1):584 (1997).
U.S. Pat. No. 4,179,337 to Davis et al. discloses that proteins coupled to PEG have enhanced blood circulation lifetime because of reduced kidney clearance and reduced immunogenicity. The lack of toxicity of the polymer and its rapid clearance from the body are advantageous for pharmaceutical applications.
To couple PEG to a molecule such as a protein or a small drug molecule, it is necessary to use an "activated derivative" of the PEG having a functional group at the terminus suitable for reaction with a group on the other molecule. For example, the hydroxyl group of CH.sub.3 O--PEG--OH can be converted to an aldehyde group, and this aldehyde group can then be covalently linked to a molecule or surface bearing one or more amine groups using the method of reductive amination. An example of this approach is described in U.S. Pat. No. 5,252,714 to Harris and Herati. The patent describes the preparation of PEG propionaldehyde, EQU OHC--CH.sub.2 CH.sub.2 --O--PEG--O--CH.sub.2 CH.sub.2 --CHO,
and its reaction in reductive amination of amine-bearing surfaces and molecules. Similarly, methoxy-PEG acetaldehyde, EQU CH.sub.3 O--PEG--O--CH.sub.2 --CHO,
was also used to link methoxy-PEG to a protein by reductive amination. See Chamow et. al. Bioconjugate Chemistry, 5:133(1994). In reductive amination the aldehyde and amine are mixed with a reducing agent, such as NaCNBH.sub.3, to provide a new amine: ##STR2##
However, as Zalipsky pointed out in reviewing activated PEGs for preparation of conjugates, the use of PEG acetaldehyde has been limited by its high reactivity, which leads to condensation side reactions. See Bioconjugate Chemistry, 6:150 (1995). In addition, PEG acetaldehyde has also proven difficult to prepare in high purity. For example, in the above-discussed reductive amination in which methoxy-PEG acetaldehyde, CH.sub.3 O--PEG--O--CH.sub.2 --CHO, was used to link methoxy-PEG to a protein, the purity of the methoxy-PEG acetaldehyde was only 52%. The impurity causes serious difficulties in forming PEG-biomolecule conjugates because of the required subsequent purification steps and loss of valuable bioactive molecules, such as proteins.
Oxidation reactions are problematic when working with aldehydes. Aldehydes in general are known to be subject to facile oxidation to form carboxylic acids. This oxidation can occur during isolation, purification, use or storage. These oxidized aldehydes typically are not desirable for conjugation with biomolecules. Oxidation results in a number of impurities and the reaction products are difficult to isolate.
Thus, the PEG aldehydes known heretofore in the art proved to be unsatisfactory for coupling PEG or its derivatives to biomolecules. The condensation and oxidation reactions substantially limit the uses of these PEG aldehydes. Therefore, there is need for improved methods of coupling PEG to biomolecules.